Mechanistic and steric issues in the oxidation of phenolic and non-phenolic compounds by laccase or laccase-mediator systems. the case of bifunctional substrates

Article abstract of DOI:10.1039/b516719a

Steric and redox issues of phenolic and non-phenolic substrates are investigated for a better insight of the reactivity features of the phenoloxidase laccase. Whenever a substrate is endowed with a redox potential too high for direct monoelectronic oxidation by the enzyme, or else is too much encumbered to access the enzymatic pocket, redox mediators overcome the problem, behaving as an interface between enzyme and substrate. For example, the small-sized mediator ABTS, once oxidised by laccase, fruitfully interacts with bulky substrates, 2,4,6-tri(Bu^t)-phenol providing a significant case. Other mediators, for example HBT, resort to a radical oxidation mechanism precluded to laccase, and can react with non-phenolic substrates which are impossible for the enzyme. The advantages provided by the mediators are discussed, and suitable phenolic compounds, as precursors of phenoxyl radical intermediates, emerge as a new proficient class. They could be the true natural mediators of laccase in the oxidative delignification. In fact, phenoxyl radical fragments generated by laccase from lignin, or from phenolic monomer residuals from the building up of lignin polymer or else deriving from lignin by oxidation with other ligninolytic enzymes, could oxidise non-phenolic residues of lignin thereby causing the breakdown of its alkyl network. The novel mechanistic probe 3,5-di(Bu^t)-4-OH-benzyl alcohol enables the decoupling of the reactivity channels of a phenolic vs. a benzylic alcohol moiety in the enzymatic oxidation of bifunctional substrates having structural features comparable to portions of lignin. Experimental support is thereby attained for the central role of laccase in biodelignification, in spite of the seemingly lower oxidation power of this enzyme with respect to other and stronger oxidising enzymes excreted by ligninolytic fungi. the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006.

Full text of DOI:10.1039/b516719a

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PAPER
www.rsc.org/njc | New Journal of Chemistry
Mechanistic and steric issues in the oxidation of phenolic and
non-phenolic compounds by laccase or laccase-mediator systems.
The case of bifunctional substrates
Francesca d’Acunzo, Carlo Galli,* Patrizia Gentili* and Federica Sergi
Received (in Montpellier, France) 25th November 2005, Accepted 30th January 2006
First published as an Advance Article on the web 21st February 2006
DOI: 10.1039/b516719a
Steric and redox issues of phenolic and non-phenolic substrates are investigated for a better
insight of the reactivity features of the phenoloxidase laccase. Whenever a substrate is endowed
with a redox potential too high for direct monoelectronic oxidation by the enzyme, or else is too
much encumbered to access the enzymatic pocket, redox mediators overcome the problem,
behaving as an interface between enzyme and substrate. For example, the small-sized mediator
ABTS, once oxidised by laccase, fruitfully interacts with bulky substrates, 2,4,6-tri(But)-phenol
providing a significant case. Other mediators, for example HBT, resort to a radical oxidation
mechanism precluded to laccase, and can react with non-phenolic substrates which are impossible
for the enzyme. The advantages provided by the mediators are discussed, and suitable phenolic
compounds, as precursors of phenoxyl radical intermediates, emerge as a new proficient class.
They could be the true natural mediators of laccase in the oxidative delignification. In fact,
phenoxyl radical fragments generated by laccase from lignin, or from phenolic monomer residuals
from the building up of lignin polymer or else deriving from lignin by oxidation with other
ligninolytic enzymes, could oxidise non-phenolic residues of lignin thereby causing the breakdown
of its alkyl network. The novel mechanistic probe 3,5-di(But)-4-OH-benzyl alcohol enables the
decoupling of the reactivity channels of a phenolic vs. a benzylic alcohol moiety in the enzymatic
oxidation of bifunctional substrates having structural features comparable to portions of lignin.
Experimental support is thereby attained for the central role of laccase in biodelignification, in
spite of the seemingly lower oxidation power of this enzyme with respect to other and stronger
oxidising enzymes excreted by ligninolytic fungi.
laccase in the biodelignification. However, suitable com-
Introduction
pounds, often referred to as redox mediators,13 enable laccase
to oxidise indirectly non-phenolic lignin units or substrates.14
Following monoelectronic oxidation of the mediator by the
enzyme, the oxidised mediator (Medox in Scheme 2) reacts
with non-phenolics, such as benzylic alcohols and ethers,
according to mechanisms unattainable to laccase, as for
example by H-atom abstraction. Several laccase/mediator
systems have been investigated, and found capable of oxidi-
zing benzylic lignin models or lignin itself at the expense of
oxygen.7,10,14–16
Laccase, a class of ‘blue copper’ oxidase excreted by basidio-
mycete fungi,1 contains four copper ions (one T1 copper and a
T2/T3 trinuclear copper cluster)1,2 and cooperates with other
enzymes in the biodelignification process.1–4 Four molecules of
a reducing substrate are oxidised by this enzyme coupled to the
four-electron reduction of oxygen to water.2,5 In view of the
low redox potential of the T1 Cu(II) site, which is in the range
of 0.5–0.8 V vs. NHE depending on the fungal source,1,2,6
laccase typically oxidises phenols (phenoloxidase activity;
Scheme 1) or phenolic lignin units, due to matching redox
features.1,7 Electron abstraction and subsequent deprotona-
tion gives rise to phenoxyl radicals that undergo oligomerisa-
tion, or cleavage of the aromatic ring upon dioxygen attack.8,9
Non-phenolic substrates, having redox potentials above
1.3 V,7a,10 are more resistant to monoelectronic oxidation
and are not oxidised by laccase directly. Consistently, lignin
is a recalcitrant phenolic polymer with respect to oxidation,1,11
because non-phenolic groups represent more than 70% of its
total residues.12 This would seem to limit the relevance of
Among the mediators, those presenting the 4N–OH moiety
(1-hydroxybenzotriazole, HBT; N-hydroxyphthalimide, HPI;
violuric acid, VLA; Scheme 3) proved very efficient towards
benzylic substrates, through a radical H-abstraction route of
oxidation involving the aminoxyl radical (4N–Oꢀ) intermedi-
ate as the Medox species.7,14,15 TEMPO, a stable 4N–Oꢀ
species, proved the most efficient mediator towards benzyl
Dipartimento di Chimica and IMC-CNR Sezione Meccanismi di
`
Reazione, Universita ‘La Sapienza’, P. le A. Moro 5, I-00185 Roma,
Italy. E-mail: carlo.galli@uniroma1.it; Fax: þ39 06 490421
Scheme 1 The redox cycle of the phenoloxidase laccase.
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Scheme 2 The redox cycle of a laccase-mediator system.
–dioxane) needed to be used to increase the solubility of
partially hydrophobic substrates, at the expense of a moderate
reduction of the enzyme activity.19 It was first necessary to
evaluate unambiguously any specific effect affecting the direct
reactivity of laccase with either a phenol or a benzyl alcohol
group. To assess the extent of oxidation in these experiments,
we monitor the consumption of the substrate, because it is
easier and less ambiguous than any quantification of oligo-
meric mixtures of products.
Scheme 3 Common non-natural mediators of laccase.
alcohols, but the oxidation mechanism is ionic and not radical
in this case.14,15,17
The quest for possible natural mediators of laccase has been
undertaken, and phenoxyl radicals have been recently sug-
gested for this role.7 Once generated from phenolic groups
through the phenoloxidase activity of the enzyme (Scheme 1),
the phenoxyl radicals could remove benzylic hydrogens from
non-phenolic residues (as much as the above 4N–Oꢀ inter-
mediates do), thereby mediating a radical oxidation of lignin
polymer. Experimental evidence supports this hypothesis,
since phenoxyl radicals from phenolphthalein or its derivatives
have been found able to oxidise benzylic model compounds by
H-abstraction,7a,18 before being depleted in competing phenol-
coupling oligomerisation (Scheme 4).
Oxidation of non-phenolics
Laccase does not react with benzyl alcohol directly,1 nor with
the mono-methoxy (e.g., p-anisyl alcohol) or 3,4-dimethoxy
(i.e., veratryl alcohol, VA) or 2,4,5-trimethoxy (only 3% of the
corresponding aldehyde being obtained) derivatives, nor with
4-MeO-ethylbenzene,20 all being model compounds of recur-
ring structural motifs in lignin.12 Basically, the half-wave
potential of these substrates (given in Table 1),10,21 which is
above 1.2 V and beyond the electron-abstraction reach of
laccase (0.78 V vs. NHE, in the specific case of Trametes villosa
laccase),6a prevents monoelectronic oxidation to occur. Ac-
cordingly, neither anisole, i.e., the methyl ether derivative of
phenol, is oxidised by laccase due to the unsuited anodic
potential (E1/2 2.0 V).10 However, the half-wave potential of
1,2,4,5-tetramethoxybenzene (E1/2 1.05 V vs. NHE) is acces-
sible to laccase, and indeed this non-phenolic is quantitatively
oxidised, as also hinted in the literature,22 whereas the posi-
tional isomer 1,2,3,5-tetramethoxybenzene is not, being the
removal of electrons more difficult in view of the higher half-
wave potential (E1/2 1.44 V vs. NHE). In conclusion any
substrate, regardless its phenolic or non-phenolic nature, is a
sound candidate for the monoelectronic oxidation by laccase,
provided that the electrochemical potential is suitable.
In this paper we have addressed the key issue of mediation
of laccase by phenoxyl radicals once more, and have expanded
the study to the oxidation of bifunctional substrates having
both a phenol and a benzyl alcohol moiety, this being a
recurring structural feature in lignin.12 One of the experimen-
tal problems we met was how to sort out unambiguously the
contribution of solubility and steric issues upon the direct
reactivity of laccase toward the bifunctional substrates, with
or without mediators. A central role of laccase in biodeligni-
fication emerges from the present study.
Results and discussion
As anticipated in the Introduction, recalcitrant non-pheno-
lic substrates can be oxidised if laccase is used together with
4N–OH mediators (Scheme 3 and Table 1) which, in view of
their pKa values,7a are extensively deprotonated (4N–Oꢂ) in
solution at pH 5. The radical route delineated in Scheme 5
operates through the intervention of the aminoxyl radical
(4N–Oꢀ) as the active Medox species.14
Laccase from the fungus Trametes villosa (viz. Poliporus
pinsitus) was employed. The oxidation reactions were per-
formed at room temperature in buffered (0.1 M sodium citrate,
pH 5) water solution, initially purged with O2 before the
addition of the enzyme. In many cases a mixed solvent (water
Redox features of the substrate have no major importance
upon a radical H-atom transfer (HAT) route, where only
the enthalpic balance between the breaking and forming of
bonds is relevant.7a For many 4N–Oꢀ species the energy of
the NO–H bond being formed on H-abstraction (BDEOH
values in the 78–88 kcal molꢂ1 range)7a matches that of the
scissile benzylic C–H bond in the substrate.23 Mediator TEM-
PO, instead, undertakes an ionic route of oxidation of benzylic
Scheme 4 Partition of a phenoxyl radical between H-abstraction
from a non-phenolic substrate and coupling with another phenolic
precursor.
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Table 1 Recovery of substrate, or extent of oxidation, in reaction of phenolic or non-phenolic substrates with laccase or laccase/mediator
systems, for 24 h at 25 1Ca
Recovered substrate Recovered substrate Oxidized product Oxidized product
(%) with laccase
in water
Esub1/2, in V vs.
NHE (in H2O)
(%) with laccase in
a mixed solventc
(%) with laccase/ (%) with
TEMPO
b
Substrate
laccase/HBT
Benzyl alcohol
p-Anisyl alcohol
VA
2.68 (in MeCN)
1.98 (in MeCN)
1.36
99d
99d
98d
95d
—
—
—
—
92e
98e
99e
85e
o1cd
—
—
—
—
—
30e
76e
92e
—
2,4,5-Trimethoxy-benzyl alcohol ca. 1.2
60g
99d
o1fi
90
4-MeO-ethylbenzene
1,2,4,5-Tetra-methoxybenzene
1,2,3,5-Tetra-methoxybenzene
Anisole
Phenol
4-But-phenol
ca. 1.7 (in MeCN)
1.05 (in MeCN)
1.44 (in MeCN)
2.0 (in MeCN)
0.87
0.82
0.87
1.01
0.71
28ch
—
—
—
—
—
—
—
99d
o1f
o1f
98d
95d
99d
—
—
—
b-Naphthol
o1f
o1fi
99d
b
—
—
—
—
2,4,6-Trichlorophenol
2,4,6-Tri(But)-phenol
a
o1cd
40cj
Conditions: [substrate] 20 mM, 10 U of laccase; [mediator] 6 mM, if employed. From ref. 10 and 21; in general, run at 25 1C, acid pH and
c
d
e
f
0.1 V sꢂ1
is recovered. 2,4,5-Trimethoxybenzaldehyde is detected (3%), in addition to a dimeric derivative formed by acid catalysis (see Experimental).
. In H2O : dioxane 1 : 1. Quantitative recovery of the substrate. The product is the corresponding aldehyde. No substrate
g
h
i
As a mixture of 4-MeO-acetophenone and 1-(4-MeO-phenyl)ethanol (from ref. 20). Minor amounts of quinone are detected by GC-MS. 10%
j
of di(But)quinone is also detected by GC-MS (see Experimental).
alcohols to aldehydes through the oxoammonium ion (4NQ
O1) acting as the Medox species (Scheme 6).7a,14,15,17
Table 1 shows that the functional group of benzyl alcohols,
which is unreactive with laccase, is susceptible to oxidation by
the specific Medox form (either 4N–Oꢀ or 4NQO1) of
suitable mediators through mechanisms other than the mono-
electronic one of the enzyme. Finally, oxidation of an alkyl-
arene, such as 4-MeO-ethylbenzene, takes place appreciably
through the HAT route with laccase/HBT, and solubility
problems can be overcome by the use of mixed solvents.20
The ionic route of TEMPO, in contrast, does not operate with
the alkylarenes, the necessary requirement of a nucleophilic
attack onto TEMPO-oxoammonium being unfeasible for this
kind of substrate, while possible for the lone-pair of benzyl
alcohols.17,20
to-nucleus or nucleus-to-nucleus couplings.24 The same occurs
with a more hydrophobic phenol such as 4-tert-butyl-phenol;
however b-naphthol, 2,4,6-trichlorophenol and 2,4,6-tri(But)-
phenol, in spite of the appropriate redox potential value, are
not oxidised by laccase. There is certainly an effect of a
reduced (or lack of) solubility in water, because b-naphthol
and 2,4,6-trichlorophenol are fully consumed whenever the
reaction is run in a water : dioxane (1 : 1) mixed solvent, but
this is insufficient for 2,4,6-tri(But)phenol which continues to
be unreactive. Here steric factors may hinder the approach of
the substrate to the enzyme active site, and consequently
preclude the oxidation. Not all the phenols are therefore fitting
substrates for the phenoloxidase laccase! This steric problem is
solved by suitable mediators. The 4N–Oꢀ species generated
from HBT by laccase, in view of its small size and matching
value of BDE (Scheme 7),7a abstracts H-atom from the O–H
bond of 2,4,6-tri(But)phenol and affords the corresponding
phenoxyl radical. The latter, having the 2,4,6-positions hin-
dered, undergoes preferential loss of t-Buꢀ to give di-t-butyl-p-
benzoquinone (10%), besides oligomeric products from
Oxidation of phenols
Laccase promotes the monoelectronic oxidation of phenols
because the redox potential of phenolic substrates (0.5–0.9 V,
at acid pH)21 and enzyme (0.6–0.8 V)1,2,6 is well matched; this
allows the transfer of electrons and ensuing quantitative
depletion of the substrate, as reported in Table 1 for phenol
itself. Phenoxyl-to-phenol oligomers are produced via oxygen-
Scheme 6 The ionic route of oxidation of benzyl alcohols by laccase/
TEMPO.
Scheme 5 The radical H-atom transfer (HAT) route of oxidation of a
non-phenolic substrate by laccase and 4N–OH mediators.
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of reactivity reported in Table 2. The structurally complex
phenol 1 is soluble in water and less encumbered than 2,4,6-
tri(But)phenol, in view of the smaller size of the CH2OH
groups in the ortho position. Consistently, it can approach
the enzyme and is quantitatively oxidised monoelectronically
to oligomeric and ring-cleavage products. Mediation of lac-
case would consequently appear unnecessary but, whenever
TEMPO is added, an important change in the product mixture
is found. The intermediate phenoxyl radical of 1, resulting
from interaction with laccase, is trapped by TEMPO (a radical
scavenger),25 and both the phenol-coupling and ring-cleavage
pathways are accordingly put off. Only the oxidation of the
hydroxymethyl groups of 1 into formyl-groups is observed,26
in keeping with similar transformations of benzyl alcohol
groups by TEMPO-oxoammonium (Scheme 6).17 Therefore,
TEMPO intercepts and stops the normal reactivity pattern of
laccase towards the ‘phenolic part’ of 1, while the enzyme-
produced TEMPO-oxoammonium oxidises the non-phenolic
side-chain of 1 regioselectively. With the trimeric derivative of
1 (i.e., 2), lack of solubility in water hampers the direct
oxidation by laccase, this being even more pronounced with
tetrameric derivative 3. Use of the mixed solvent improves the
situation with 2, but not with 3. Oxidation of the latter takes
place extensively with the laccase/TEMPO system but, once
again, it is specifically directed towards the CH2OH side-
chain.26 Besides, monoelectronic oxidation of 3 takes place
Scheme 7 Thermochemical balance for the radical HAT mechanism
between a 4N–Oꢀ intermediate and a phenol acting as a H-donor
substrate.
oxygen-to-nucleus couplings,24d followed by loss of t-Buꢀ. No
oxidation of 2,4,6-tri(But)phenol takes place instead with lac-
case/TEMPO, because the ionic route through TEMPO-
oxoammonium is structurally precluded to a phenol.
Depletion of 2,4,6-tri(But)phenol with laccase occurs more
significantly (i.e., 55%) in the presence of Phenol Red (Table
2). This particular phenol, once monoelectronically oxidised
by laccase to a relatively stable phenoxyl radical,7a,18 performs
as a mediator by efficiently abstracting the H-atom from the
O–H bond of 2,4,6-tri(But)phenol. The subsequent reactivity
channels of the 2,4,6-tri(But)phenoxyl radical described above
with the N–OH mediator (cf. Scheme 7) then become acces-
sible. Finally, 2,20-azinobis-(3-ethylbenzothiazoline-6-sulfo-
nate) (viz. ABTS)7a,10,14,15 mediates laccase in an even more
extensive (90%) oxidative degradation of 2,4,6-tri(But)phenol.
The Medox form of ABTS (i.e., ABTSꢀ1) is in fact endowed
with a redox potential (0.69 V)10 suitable to monoelectronic
oxidation of the phenolic substrate (0.71 V), the subsequent
depletion of which occurs by loss of t-Buꢀ to give di-t-butyl-p-
benzoquinone (30%) besides oligomeric products. Therefore,
the oxidative degradation of the recalcitrant 2,4,6-tri(But)phe-
nol takes place appreciably either by the intervention of
radical mediators according to the HAT route or, and more
extensively, by electron transfer to ABTSꢀ1. In both ways the
steric incompatibility of the encumbered phenol towards the
active site of laccase, which hampered a direct oxidation, is
overcome.
Comparable is the steric bulk of 2,6-di(But)-4-Me-phenol
and 2,6-di(But)-4-MeO-phenol, or that of the disubstituted
2,6-di(But)phenol, which are largely recovered unreacted on
exposure to laccase (Table 2). One concludes that sterically
demanding groups ortho to the phenolic OH prevent the
approach of the substrate to the enzyme active site, and any
ensuing oxidation. In keeping with this explanation 2,4-di
(But)phenol, having only one encumbered ortho position, is
partially oxidised by laccase (25%) and a dimeric coupling
product (ca. 10%) detected.
1
quantitatively with laccase/ABTS through the ABTSꢀ inter-
mediate, as also observed for the encumbered 2,4,6-tri(But)-
phenol. This stresses the importance of small-sized diffusible
mediators for an efficient oxidation of bulky substrates, in-
cluding phenolic ones.
Phenols as radical mediators of laccase towards non-phenolics
Structural features make the phenoxyl radical from Phenol
Red sufficiently stable and ‘slow’ in self-coupling routes,18 so
to enable H-abstraction (cf. Scheme 4). In fact Phenol Red
mediates laccase efficiently in the radical oxidation of the
encumbered 2,4,6-tri(But)phenol (in Table 2). Taking advan-
tage from this long-living phenoxyl radical, the laccase/Phenol
Red system has been exploited for the benchmark oxidation of
p-anisyl alcohol, the most simple lignin model compound, into
the corresponding aldehyde in reasonable yield (Table 3).7a
Inspired by this result, we searched for any ability of the
encumbered phenoxyl radical from 2,4,6-tri(But)phenol to
mediate similarly, but no significant oxidation of p-anisyl
alcohol with laccase and 2,4,6-tri(But)phenol was obtained.
This may simply confirm the inability of laccase to convert this
phenol into the phenoxyl radical for steric reasons. Conver-
sely, catechol (1,2-dihydroxybenzene) compares well with
Phenol Red because it mediates laccase in the oxidation of
p-anisyl alcohol appreciably. Ortho-diphenols are recurring
structural motifs in lignin and, whenever sufficiently stable as
phenoxyl radicals, they could perform as natural mediators of
laccase towards non-phenolic and phenolic units. Similarly
effective is the 1,2,3-trihydroxy-analogue pyrogallol, and even
more so hydroquinone, but not 2,6-(MeO)2-phenol, which
sometimes is used to assay the activity of laccase.27 Neither
barbituric nor uric acids, which are natural compounds
The size of the 2,6 substituents must be large enough to
hinder the approach to the active site, as indicated by the
quantitative oxidation of the smaller 2,4,6-trimethylphenol by
laccase (only 1% recovered; cf. also 2,4,6-trichlorophenol in
Table 1), to give oligomeric products plus 4-hydroxy-3,5-
dimethylbenzaldehyde (20%). Significant also is another trend
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Table 2 Consumption of encumbered phenols, and evidence of product(s), in the aerobic oxidations with laccase or laccase/mediator systems, at
25 1Ca
Recovered
substrate
(%) with
Recovered
Recovered
substrate (%)
with laccase/
ABTS
Recovered
substrate (%)
with laccase/
TEMPO
Recovered
substrate (%)
with laccase
substrate (%)
with laccase/
Phenol Red
Substrate
laccase/HBT
2,4,6-Tri(But)phenol
99
99
60; 10% quinone 45
10; 30% quinone 99
47; 1% 4-CH2OH, 2; 2% 4-CHO, —
2,6-Di(But)-4-Me-phenol
90; 2% quinone,
tr. 4-CH2OH
82; 13% quinone
9% quinone
13% quinone,
1; 75% quinone
2,6-Di(But)-4-MeO-phenol
2,6-Di(But)phenol
93; 4% quinone
94
75; 10% dimer
1; 20% aldehyde
—
—
—
—
—
—
—
99
—
—
—
—
—
—
2,4-Di(But)phenol
2,4,6-Tri(Me)phenol
o1b
—
—
—
o1 (but
different
products)
82bc
40
—
—
—
5
99d
—
o1
13
a
b
c
d
Conditions as in Table 1, in H2O : dioxane 1 : 1. In buffered water. Becomes 45% in the mixed solvent. Both in buffered water and in the
mixed solvent.
structurally related to the proficient mediator VLA,7a appear
to mediate laccase toward p-anisyl alcohol.
Additional examples of aryloxyl radicals, which are suffi-
ciently stable to play an essential role for the hosting protein,
are known and found in the ribonucleotide reductase, galac-
tose oxidase and prostaglandine H synthase enzymes, besides
photosystem II.28
Oxidation of bifunctional substrates
Scheme 8 Conceivable functionalisation of a side-chain in para to a
phenoxyl radical.
Anodic oxidation of phenols bearing suitable substituents in
para, such as methyl groups, is known to give rise to quinone-
methide and dimerisation products.29 Because the presence of
both a phenol and a benzyl alcohol (or other benzylic groups)
on the same aromatic ring is widespread in lignin,12 by the
same token any laccase-induced monoelectronic oxidation of
the phenolic group in a bifunctional substrate could ‘shift’
intramolecularly from the phenoxyl radical to the benzylic
center (Scheme 8).30 This would enable subsequent transfor-
mation/oxidation of the latter, as much as any mediated
Table 3 Yield of p-anisaldehyde from the aerobic benchmark oxidation of p-anisyl alcohol with laccase and phenolic or phenolic-like mediators
% aldehyde
% aldehyde
with laccase/
Phenol Red
with laccase/
2,4,6-tri(But)
phenol
% aldehyde
with laccase/
catechol
% aldehyde
with laccase/
hydroquinone
% aldehyde
with laccase/
pyrogallol
% aldehyde
with laccase/2,6-
(MeO)2C6H3OH
% aldehyde
with laccase/
barbituric acid
Substrate
p-Anisyl
alcohol
20 (35, after 48 h)
o2
19
32
18
r1
r1
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Table 4 Recovery of bifunctional substrates (having both phenol and benzyl alcohol moieties) under aerobic oxidation with laccase or laccase/
mediator systems, in H2O : dioxane 1 : 1 at 25 1C
% recovery
with laccase
% recovery with
laccase/ABTS
% recovery with
laccase/TEMPO
% recovery
with laccase/HBT
% recovery
with laccase/HPI
Substrate
3,5-Di(But)-4-OH-benzyl
alcohol (4)
95
99
0; o1% of 5
0; 100% of 5
1; 29% of 5
3,5-Di(But)-4-OMe-benzyl
alcohol (6)
2,4,6-Tri(Me)phenol
95
—
—
—
99
—
83; 17% of 7
93; 7% of 7
1; 20%
aldehyde
97
—
—
—
—
2,4,6-Tri(Me)-anisole
radical oxidation regiospecifically directed towards the
benzylic group would do.
Bulky substituents in ortho to free phenolic groups in lignin,
while blocking (or retarding) the normal coupling channels of
the intermediate phenoxyl radical (cf. Table 2),2a could par-
tially drive the reactivity toward the conjugated benzylic
center; this would induce a Ca–Cb cleavage of the alkyl chains
in lignin, as already documented,31 even on suitable arylalkyl
models which yield aromatic aldehydes as chain-cleaved pro-
ducts.15a,32 Therefore, a depolymerisation of lignin without the
need for external/added mediators would ensue. This endorses
the function of phenols not only as ‘natural’ substrates but
also as natural mediators of laccase, supporting a central role
of the latter in biodelignification. Consistently, the monoelec-
tronic oxidation of hindered phenols, such as 2,6-di(But)-4-
methylphenol (and others), by DDQ (2,3-dichloro-5,6-dicya-
no-1,4-benzoquinone) has been already described.33 The for-
mation of dimers via C–C or C–O coupling, subsequent
dissociation into radicals and further disproportionation to a
quinone–methide, evolving in the formation of 3,5-di(But)-4-
hydroxybenzaldehyde as a benzylic oxidation product, are
documented.
Scheme 9 Reaction pathways with probe substrate 4.
opening products, which escaped our GC detection being
either high-boiling or water-soluble, respectively), made avail-
able by this small-sized Medox species. No evidence for
participation from the ‘spectator’ CH2OH group in para to
the overall reactivity is obtained, since less than 1% of 3,5-
di(But)-4-hydroxy-benzaldehyde (5) is detected, i.e. the specific
oxidation product of the benzylic moiety. The oxidation of 4
with laccase/TEMPO gives an almost opposite outcome (Ta-
ble 4). Full consumption of 4 is now accompanied by quanti-
tative and regioselective conversion into aldehyde 5, consistent
with the operation of the ionic mechanism of oxidation of the
benzyl alcohol moiety by TEMPO-oxoammonium. Finally,
the intervention of the radical Medox species 4N–Oꢀ from the
use of laccase/HBT promotes the H-abstraction route, and
leads to the consumption of 4 with a significant formation
(29%) of 5. Does the latter derive from direct H-abstraction
from the benzylic C–H bond of the CH2OH group, or from
initial H-abstraction from the phenolic O–H followed by the
resonance/dehydrogenation route outlined in Scheme 8?
In order to solve this mechanistic conundrum, conversion of
the phenolic group of 4 into a methoxy derivative, i.e. 3,5-
di(But)-4-OMe-benzyl alcohol (6), was devised. In this case,
any HAT route (cf. Scheme 5) could occur only on the
CH2OH group, the expected product being 3,5-di(But)-4-
OMe-benzaldehyde (7) (Scheme 10). This strategy was in-
spired by finding that, whereas 2,4,6-trimethylphenol reacts
A problem of clear interpretation of the results in bifunc-
tional substrates arises, however. In fact, the reactivity of the
phenolic group that reacts with laccase directly, as well as the
reactivity of the benzylic group that does not react in the
absence of mediators, are somewhat ‘geared’ (see Experimen-
tal). For example, it is quite obvious that a simple bifunctional
substrate such as 4-hydroxy-benzyl alcohol reacts with laccase
quantitatively, being a phenol. However, this is not sufficient
indication for any participation of the benzylic moiety to
overall reactivity through the operation of the delocalisation/
dehydrogenation (or disproportionation)33 pathway depend-
ing on the phenoxyl radical and quinone-methide, as deli-
neated in Scheme 8. In order to unambiguously decouple the
reactivity channels of the two functional groups, appropriate
substrates ought to be used.
In this context, 3,5-di(But)-4-hydroxy-benzyl alcohol (4)
becomes a significant mechanistic probe (Scheme 9). It is
recovered 95% unchanged by exposure to laccase (Table 4),
in keeping with the lack of reactivity of 2,4,6-encumbered
phenols (Table 2). However, phenol 4 substantially gains in
reactivity under the presence of mediators. Monoelectronic
oxidation via ABTSꢀ1, generated from ABTS by laccase, takes
place as it did in the case of 2,4,6-tri(But)phenol (Table 2),
leading to full consumption of 4 through the typical reactivity
channels of phenolic oxidations (i.e., oligomers and ring-
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is sufficiently low, 1,2,4,5-tetramethoxybenzene being a fitting
example. Quite the opposite, solubility or steric issues make
the oxidation of specific phenolic substrates unattainable to
laccase, 2,4,6-tri(But)-phenol providing a significant case. In
this event, redox mediators enable the enzyme to solve the
problem, because the oxidised mediator acts as the interface
between laccase and the substrate. Small-sized mediators can
fruitfully interact with encumbered substrates that cannot
access the enzymatic pocket directly. On the other hand,
substrates endowed with high electrochemical potential can
be oxidised by radical mediators through the operation of a H-
abstraction mechanism inaccessible to the enzyme itself. On
this topic we stress the importance of phenoxyl radicals. They
could be the true natural mediators of laccase for the oxidative
degradation of the aromatic polymer lignin, where phenolic
but mostly non-phenolic (benzylic) residues are present. Phe-
noxyl radicals produced by the phenoloxidase activity of
laccase either from lignin, from phenolic monomers residuals
from the building up of lignin polymer,34 or deriving from
lignin by oxidation with other ligninolytic enzymes,1 provided
that they are sufficiently long-living, could mediate laccase
towards the oxidation of non-phenolic lignin units. Steric
effects could grant this long-living feature to appropriate
phenoxyl radicals, enabling them to perform as radical me-
diators before being depleted in competing oligomerisation
pathways.7a,18,35 In keeping with this point, the structural
peculiarity of the aromatic groups of lignin, where both
phenolic and benzylic moieties are often present together as
substituents, is remarked upon and 3,5-di(But)-4-OH-benzyl
alcohol exploited as a significant mechanistic probe. The
experimental information acquired reveals that initial forma-
tion of a phenoxyl radical on the one side of the aromatic
bifunctional molecule might activate the benzylic substituent
on the other side. By extrapolation, the geared reactivity of
these two kinds of functional groups could induce the break-
down of the alkyl network of the polymer lignin under oxygen-
assisted radical routes, enabling its degradation. In conclusion,
the experimental evidence presented here supports the relevant
role of laccase in the biodelignification, despite the seemingly
lower power of this enzyme with respect to other and stronger
oxidising enzymes produced by ligninolytic fungi, such as
Lignin peroxidase or manganese peroxidase.1
Scheme 10 In search of a genuine phenol-dependent benzylic func-
tionalisation.
quantitatively with laccase, 2,4,6-trimethylanisole does not do
so and is recovered quantitatively (Table 4). Obviously, con-
version of the phenolic into a methyl ether group, in 2,4,6-
trimethylanisole, by enhancing the redox potential of the
substrate (compare anisole vs. phenol in Table 1), hampered
the ‘normal’ phenoloxidase route (to oligomeric coupling and
ring-cleavage products) and substrate depletion. But it pre-
vented also any oxidation of the methyl group(s) of 2,4,6-
trimethylanisole into formyl moiety, at variance with the
partial (20%) oxidation of the 4-methyl group that is observed
for 2,4,6-trimethylphenol. It is therefore likely that the latter
oxidation is a phenol-dependent side-chain functionalisation,
in keeping with Scheme 8. Consistent with this observation
and with our expectation, oxidation of the ether 6 by laccase/
HBT gave 17% of aldehyde 7, more than 80% of 6 being
recovered unreacted. If such an extent of formation of 7 may
be taken as the intrinsic reactivity of the CH2OH group of 6 in
the HAT route, it supports our claim that the more abundant
formation (29%, in Scheme 9) of aldehyde 5 in the corre-
sponding HAT route of oxidation of 4 by laccase/HBT truly
testifies for the phenoxyl-dependent side-chain functionalisa-
tion in para outlined in Scheme 8.
Experimental
General remarks
NMR spectra were taken on an AC 300 Bruker instrument. A
VARIAN CP 3800 GC, fitted with a 30 m ꢃ 0.25 mm methyl
silicone gum capillary column (CPSil5CB), was employed in
the analyses. The identity of the products was confirmed by
GC-MS analyses, run on a HP 5892 GC, equipped with a
30 m ꢃ 0.2 mm methyl silicone gum capillary column, and
coupled to a HP 5972 MSD instrument, operating at 70 eV.
An Agilent HPLC system (pump, degasser, UV-Vis detector,
and solvent delivery system), equipped with a Supelcosil LC-
18-DB 25 cm ꢃ 4.6 mm column, was used, as well as a Perkin
Elmer Sciex API 365 liquid-mass spectrometer.
Conclusions
Assessment of product yields or extent of consumption of the
substrate provide a way to monitor reactivity in the mono-
electronic oxidation reaction by laccase. The importance of the
redox potential of the substrate is stressed. Phenols, having
electrochemical potential comparable to that of the enzyme,
are typical natural substrates, whereas structurally different
compounds, endowed with substantially higher electrochemi-
cal potential, are not. However, laccase can directly oxidase a
non-phenolic substrate whenever its electrochemical potential
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Materials
meric coupling products are expected;8,9 in a few instances,
evidence for these was qualitatively obtained through LC-MS
analyses.26 In the oxidation of 2,4,6-trichlorophenol with lac-
case, minor amounts of dichloroquinone were detected from
GC-MS (m/z 176). In the oxidation of 2,4,6-tri(But)phenol, the
formation of di(But)quinone was inferred from GC-MS ana-
lysis (m/z 220); it was possible to isolate it by a short-path
column of silica gel (hexane : diethyl ether 30 : 1 as the
eluent) from the crude material of the oxidation with laccase/
HBT; it has a mp 66–68 1C. 1H-NMR d (CDCl3): 6.49 (s, 2H,
CQC–H), 1.27 (s, 18H, C(CH3)3); 13C-NMR d (CDCl3): 189.7
(CQO), 188.3 (CQO), 158.5 (Me3CCQCH), 130.7 (Me3CCQ
CH), 36.2 (CMe3), 29.9 (CH3)3. In the oxidation of 2,4,5-
trimethoxybenzyl alcohol with laccase, a dimeric compound
(m/z 348) was detected by GC-MS. It possibly originates from
the benzyl alcohol precursor by proton-induced loss of water,
followed by attack of the resulting carbocation onto a second
molecule of precursor, and loss of formaldehyde.
Most of the mediators and substrates were commercially
available (Aldrich), or already present in the laborator-
y.7a,10,15,17 Phenols 1, 2 and 3 were available from a previous
investigation.26 Buffers were prepared using ultrapure water
obtained from a MilliQ apparatus.
Synthesis of 3,5-di(But)-4-OMe-benzyl alcohol (6). A direct
conversion of the commercially available 3,5-di(But)-4-OH-
benzyl alcohol (Aldrich) into the title compound, by methyla-
tion of the phenolic hydroxyl group with MeI and KOH in
MeCN, proved unfeasible because it led to polymeric material.
This confirms the mutual interaction of the OH and CH2OH
groups in the para hypothesised in Scheme 8. Commercially
available 3,5-di(But)-4-OH-benzaldehyde (Aldrich) was in-
stead successfully methylated with a fourfold molar amount
of MeI in MeCN solution at 80 1C for 36 h, using t-BuOK as
the base. The obtained 3,5-di(But)-4-OMe-benzaldehyde (m/z
248) was quickly purified by short-path column chromatogra-
phy on silica gel with hexane : diethyl ether 30 : 1 as the
eluent, and reduced with NaBH4 in 2-propanol containing
NaOH. Product 6 was obtained in a 47% overall yield, and
analogously purified by column chromatography, to a solid
HPLC analyses
With substrates 1–3, consumption after 24 h reaction time was
determined by HPLC. The analyses were carried out with
gradients of water–methanol–isopropanol mixtures, contain-
ing 0.03% trifluoroacetic acid, at 0.5–1 mL minꢂ1 flow rate.
Quantitation of unreacted substrate was achieved by using
2-bromonaphthalene (Aldrich) as the internal standard. The
standard was added to the reaction crude, which was then
diluted in the mobile phase and filtered through 0.2 mm Teflon
syringe filters (Superchrom Varisep) prior to analysis. Liquid
mass spectrometry (LC-MS) analyses were carried out using a
triple quadrupole Perkin Elmer Sciex API 365 spectrometer
with a turbo-ion spray interface. Samples were diluted in
HPLC-grade methanol, filtered through 0.2 mm Teflon syringe
filters, and directly injected into the ion spray chamber without
chromatographic separation.
1
(m/z 250) melting at 94–96 1C. H-NMR d (CDCl3): 7.26 and
7.24 (s, 2H, ArH), 4.62 (s, 2H, CH2OH), 3.69 (s, 3H, OCH3),
1.75 (s, 1H, OH), 1.43 (s, 18H, 2 ꢃ CMe3); 13C-NMR d
(CDCl3): 159.3 (CAr–OMe), 144.0 (CAr–CH2OH), 134.9
(CAr–CMe3), 127–125 (ArH), 66.0 (ArOCH3), 64.3 (CH2OH),
35.9 (CMe3), 32.2 (Me).
Enzyme purification. Laccase from a strain of Trametes
villosa (viz. Poliporus pinsitus) (Novo Nordisk Biotech) was
employed. It was purified by ion-exchange chromatography
on Q-Sepharose by elution with phosphate buffer, and laccase
fractions having an absorption ratio A280/A610 of 20–30 were
considered sufficiently pure.7a,26 The collected fractions were
concentrated in cellulose membrane tubing (Sigma) by dialysis
against poly(ethylene glycol), a final activity of 5000 U mLꢂ1
being determined spectrophotometrically by the standard
assay with ABTS.36
Acknowledgements
We thank the Italian MIUR for financial support, as COFIN-
2004 (2004038243_002), FIRB-2001 (RBAU01PPHX) and
FIRB-2003 (RBNE034XSW) grants, and the University of
Roma ‘La Sapienza’ for the C26A043907 grant.
Enzymatic reactions
The aerobic oxidation reactions were performed at room
temperature in stirred water solution (3 mL), buffered at pH
5 (0.1 M in sodium citrate) and purged with O2 for 30 min
prior to the addition of the reagents.13,14 In general, the initial
concentration of the reagents was: [substrate] 20 mM, 3 U
mLꢂ1 of laccase, besides [mediator] 6 mM, when employed.
The reaction time was 24 h at room temperature, an atmo-
sphere of oxygen being kept in the reaction vessel by means of
a hemi-inflated latex balloon. In many cases, a 1 : 1 buffered
water : dioxane mixed solvent (3 mL) was employed to ensure
full solubility of the substrates. The reaction mixture was
extracted with ethyl acetate, dried over Na2SO4 and charac-
terised by GC and GC-MS analyses. The recovery of substrate
or the yield of oxidation product was reckoned by GC analysis
with respect to an internal standard (acetophenone or 4-
methoxyacetophenone or 4-methylbenzophenone), using sui-
table response factors. With simple phenolic substrates, oligo-
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